PROJECT SUMMARY Antigen receptor-induced calcium (Ca2+) signals regulate lymphocyte development and effector functions, but major gaps persist in our understanding of the dynamics of these signals, their transcriptional consequences and how cell fates are governed by distinct receptor-induced Ca2+ waveforms. Supporting data in this application and a pending publication reveal entirely novel and indispensable mechanisms by which Ca2+ specifically tunes TCR, but not TNF, induced NF-?B p65 and c-Rel activation. Our findings that p65 and c-Rel phosphorylation may serve as Ca2+ dependent checkpoints provide a framework for understanding how distinct patterns of Ca2+ signaling in vivo control the transcriptional programs that drive lymphocyte development and function. Central to TCR-induced Ca2+ entry are the ER transmembrane Ca2+ sensing STIM1 and STIM2 proteins, and the STIM-activated Ca2+ channel CRAC/Orai. Mice whose T cell progenitors lack STIM1/2 exhibit a selective defect in nTreg induction and the immune phenotype of these mice is virtually indistinguishable from that of mice with a c-Rel deficiency. Moreover, as nTreg development is driven by CD28 costimulation in conjunction with high affinity/avidity TCR ligation, and we have previously shown that CD28 biases this deterministic signal toward high amplitude Ca2+ spikes over a range of antigen receptor ligation avidities, we hypothesize that CD28 tuning of TCR induced Ca2+ dynamics generates a quantitatively unique waveform that drives c-Rel-dependent nTreg generation. Based on our pending publication and new supporting data, we hypothesize that TCR induced Ca2+-dependent phosphorylation of individual p65 and c-Rel Ser/Thr residues is regulated by distinct Ca2+ thresholds and waveforms, thereby providing a mechanism by which quantitatively distinct Ca2+ signals differentially regulate NF-?B-dependent gene expression controlling lymphocyte development and functions. To address this hypothesis, we will: 1) Define the nature and function of Ca2+- regulated phosphorylation of p65 and c-Rel, 2) determine how Ca2+ controls NF-?B-driven transcriptional specificity, and 3) define the Ca2+ dependent mechanisms of thymic nTreg development. We will use a custom built ?Ca2+ clamp? perfusion system to impose variations in the Ca2+ amplitude, pulse width, frequency, and total input to define how Ca2+ waveforms control sequential checkpoints in NF-?B activation and long-term imaging measurements utilizing a novel genetically encoded Ca2+ indicator (GCaMP6f) to determine how antigen-induced Ca2+ signals contribute to the regulation of nTreg development. The combined expertise of the team, the systems and approaches, and the quantitative tools developed for these studies will provide a new and detailed mechanistic understanding of how calcium dependent phosphorylation regulates transcriptional specificity of NF-?B in T cells and will provide insights into strategies and therapeutic targets for enhancing insufficient, suppressing auto-reactive, or redirecting inappropriate immune responses.